Impact of nitrogen fertilizer type and application rate on growth, nitrate accumulation, and postharvest quality of spinach

Background A balanced supply of nitrogen is essential for spinach, supporting both optimal growth and appropriate nitrate (NO3−) levels for improved storage quality. Thus, choosing the correct nitrogen fertilizer type and application rate is key for successful spinach cultivation. This study investigated the effects of different nitrogen (N) fertilizer type and application rates on the growth, nitrate content, and storage quality of spinach plants. Methods Four fertilizer types were applied at five N doses (25, 50, 200, and 400 mg N kg−1) to plants grown in plastic pots at a greenhouse. The fertilizer types used in the experiment were ammonium sulphate (AS), slow-release ammonium sulphate (SRAS), calcium nitrate (CN), and yeast residue (YR). Spinach parameters like Soil Plant Analysis Development (SPAD) values (chlorophyll content), plant height, and fresh weight were measured. Nitrate content in leaves was analyzed after storage periods simulating post-harvest handling (0, 5, and 10 days). Results The application of nitrogen fertilizer significantly influenced spinach growth parameters and nitrate content. The YRx400 treatment yielded the largest leaves (10.3 ± 0.5 cm long, 5.3 ± 0.2 cm wide). SPAD values increased with higher N doses for AS, SRAS, and CN fertilizers, with AS×400 (58.1 ± 0.8) and SRAS×400 (62.0 ± 5.8) reaching the highest values. YR treatments showed a moderate SPAD increase. Fresh weight response depended on fertilizer type, N dose, and storage period. While fresh weight increased in all fertilizers till 200 mg kg−1 dose, a decrease was observed at the highest dose for AS and CN. SRAS exhibited a more gradual increase in fresh weight with increasing nitrogen dose, without the negative impact seen at the highest dose in AS and CN. Nitrate content in spinach leaves varied by fertilizer type, dose, and storage day. CNx400 resulted in the highest NO3− content (4,395 mg kg−1) at harvest (Day 0), exceeding the European Union’s safety limit. This level decreased over 10 days of storage but remained above the limit for CN on Days 0 and 5. SRAS and YR fertilizers generally had lower NO3− concentrations throughout the experiment. Storage at +4 °C significantly affected NO3− content. While levels remained relatively stable during the first 5 days, a substantial decrease was observed by Day 10 for all fertilizers and doses, providing insights into the spinach’s nitrate content over a 10-day storage period. Conclusion For rapid early growth and potentially higher yields, AS may be suitable at moderate doses (200 mg kg−1). SRAS offers a more balanced approach, promoting sustained growth while potentially reducing NO3− accumulation compared to AS. Yeast residue, with its slow nitrogen release and consistently low NO3− levels, could be a viable option for organic spinach production.

the experiment.The plant material used was the Matador spinach (Spinacia oleracea L.) variety, which is a broad-leaved variety.This spinach variety, characterized by rapid development, large and short-petioled dark green leaves with smooth texture and oval tips, exhibiting a spreading growth habit.Additionally, it boasts high productivity and cold tolerance, making it suitable for cultivation throughout Turkiye.Ideal germination and growth occur between 15 C and 25 C.
The soil used in the experiment was obtained from the Research and Application Fields of Agricultural Faculty.The pH of the soil was 8.50, indicating alkaline conditions.Electrical conductivity was relatively low at 0.23 mmhos/cm, indicating non saline conditions.The soil had a high calcium carbonate content (29.1%) and a moderate organic matter content (1.20%).It also contained 13.4 mg P kg −1 soil, 375.2 mg K kg −1 soil, 355.2 mg Mg kg −1 soil, 1.46 mg Cu kg −1 soil, 0.55 mg Zn kg −1 soil, 6.43 mg Fe kg −1 soil, and 10.37 mg Mn kg −1 soil.

Greenhouse experiment
The experimental layout followed a completely randomized block design with three replicates, utilizing a total of 60 pots.Four different N fertilizer types with distinct characteristics were utilized in the experiment.The fertilizer types used in the experiment were ammonium sulphate (AS), slow-release ammonium sulphate (SRAS), calcium nitrate (CN), and yeast residue (YR).Five different nitrogen (N) doses (25,50,100,200, and 400 mg N kg −1 ) were applied in the form of SRAS, AS, CN, and YR.AS is the fertilizer with the highest N content (21% N), which does not contain inhibitors, organic matter, or organic carbon.CN, on the other hand, has a lower N content (11.8% N) compared to AS, and lacks inhibitors, organic matter, or organic carbon.SRAS with dicyandiamide (DCD) inhibitor contains the same 21% N as AS, but it includes a DCD inhibitor, which helps slow down the release of nitrogen.The DCD prevents the conversion of ammonium to nitrate in soil.This effect is believed to be caused by DCD binding to the active sites of ammonia monooxygenase, a copper-containing metalloenzyme crucial for ammonia-oxidizing bacteria (Amberger, 1989).This strategy enhances nitrogen use efficiency by mitigating nitrogen losses via leaching and denitrification processes.Consequently, DCD application improves fertilizer efficacy while minimizing environmental concerns such as nitrate contamination of water resources and the release of greenhouse gases.SRAS does not contain organic matter or organic carbon.YR (organic nitrogen source), has the lowest N content (3% N).It contains 35% organic matter and 16% organic carbon but does not include any inhibitors.
The experiment utilized plastic pots, each containing 2 kg of soil.The pot used in the greenhouse experiment measured 18 cm in height, 20 cm in diameter, and 18 cm in depth.As a base fertilizer application, all pots received 100 mg kg −1 phosphorus (P) in the form of KH 2 PO 4 , 125 mg kg −1 K in the form of KH 2 PO 4 , 10 mg kg −1 Fe in the form of Fe-EDTA, and 2.5 mg kg −1 Zn in the form of ZnSO 4 .The amount of sulfur in pots receiving ammonium sulfate was calculated, and CaSO 4 was applied to all pots to ensure equal sulfur content.
The experiment began with seeding (10 seeds per pot) on February 24th, 2021.Seeds germinated within approximately 8-10 days.After about 16 days (March 10th, 2021), seedlings were thinned to maintain six plants per pot.Throughout the growing season, pots were watered whenever needed to maintain soil moisture content close to field capacity, allowing for free drainage to occur.On April 16, 2021, 52-day-old plants were harvested, coinciding with the observation of significant differences in growth and development due to the varying N fertilizer types and increasing N application rates.

Measurements of spinach parameters
Soil Plant Analysis Development (SPAD) measurements were taken on a fully mature young leaf priror to harvest (52 nd day of the experiment, April 16, 2021).SPAD values were measured by averaging three readings taken from the midpoints of spinach leaves using a portable chlorophyll meter (SPAD-502 Minolta Camera Co., Tokyo, Japan) (Cordeiro, Alcantara & Barranco, 1995).Additionally, observations were conducted on five randomly chosen plants per plot for the following parameters: plant height (cm), leaf count per plant, leaf length (cm), leaf width (cm), and fresh leaf weight per plant (g).Plant height was measured as the distance from the ground to the highest point of a leaf.Leaf length was determined by measuring from the base of the petiole (leaf stalk) to the tip of the leaf blade.Leaf width was not a single measurement but the average of three widths taken across the leaf blade at 25%, 50%, and 75% of its total length.

Analysis of leaf samples
Upon harvest, six plants from each pot were carefully divided into three equal groups for separate storage periods.Two plants were allocated to each storage period to ensure balanced representation within each group.The first portion (Day 0) was washed and dried immediately in a 48 C oven.The second portion (Day 5) was washed, placed in polyethylene bags, and stored in a refrigerator (+4 C) for 5 days.Similarly, the third portion (Day 10) was washed, bagged, and refrigerated (+4 C) for 10 days.After completing the storage periods, the plant samples were dried in an oven at 70 C for 48 h for the analysis.The selection of storage durations (Day 0, Day 5, and Day 10) was carefully considered to capture the range of potential storage periods encountered by consumers.Day 0 represents the immediate post-harvest stage, reflecting the quality and nutrient content of spinach at the time of purchase.This point is particularly relevant for consumers who purchase and consume spinach within a short period.Day 5 represents an intermediate storage period, simulating situations where spinach is stored for a few days before consumption.This duration aligns with common home storage practices and provides insights into the quality changes that may occur during this period.Day 10 represents an extended storage period, mimicking scenarios where spinach is stored for a longer duration before consumption.This duration is particularly relevant for commercial storage and transportation practices, allowing for the assessment of long-term quality changes.
The selection of these storage durations is supported by research from Mudau et al. (2015), who investigated the impact of storage temperature and duration on the nutritional quality of spinach.Their findings indicate that that the concentrations of magnesium, zinc, and iron decreased after 8 days of storage at 4 C. Similarly, it is noted that samples stored at 4 C exhibited significantly higher levels of carotenoids up to 6 days, while the total phenolic compounds gradually decreased.Additionally, it is mentioned that the total antioxidant activities and vitamin C content showed a similar trend, remaining stable at 4 C but decreasing after 6 days.
The nitrate content in plant samples was measured colourimetrically using a method developed by Cataldo et al. (1975).This method relies on the formation of a yellow color complex in a strongly acidic environment.The intensity of the color complex is directly proportional to the nitrate concentration.Dried and finely ground plant samples were suspended in distilled water and incubated at 45 C for 1 h, followed by centrifugation for 15 min.A clear-colored aliquot was mixed with 5% salicylic acid in H 2 SO 4 and allowed to stand for 20 min.Then, 2 N NaOH was added while gently stirring, and the absorbance was measured at 410 nm using a spectrophotometer relative to the reference sample.The concentration of nitrates in the sample was measured by comparing it to a standard curve created using potassium nitrate (KNO₃).The results are expressed as miligrams of nitrate per gram of fresh weight of the sample.

Soil analysis
The soil used in the study was sieved through a 2 mm mesh to remove rocks and roots.Electrical conductivity (EC) and pH of soil samples were determined in 1 soil: 2.5 deionized water mixture using the method described by Rhoades (1983) Calcium carbonate content was measured by estimating the quantity of the CO 2 produced by HCl addition to the soil.Organic matter content was analysed using the Walkley-Black dichromate oxidation procedure (Nelson & Sommers, 1982).Available P was extracted with 0•5 M sodium bicarbonate (NaHCO 3 ) (Olsen et al., 1954) and determined by spectrophotometry.The available concentrations zinc (Zn), iron (Fe), manganase (Mn) and cupper (Cu) were determined after extraction with DTPA solution (Lindsay & Norvell, 1978).Available nitrogen potassium (K) and magnesium (Mg) contents in soil were determined from the neutral 1 mol/L ammonium acetate extracts (1:5, m/V) and measured by a flame Photometer (Knudsen, Peterson & Pratt, 1982).

Statistical analysis
All measured variables were subjected to statistical analysis using the SPSS software package.A variance analysis (ANOVA) was conducted to assess the presence of statistically significant differences among the means of treatment groups.In the experiment, four N fertilizer types, five N doses, and three different storage periods were included as the factors.The effects of individual factors, as well as the effects of two-way and three-way interactions, were analyzed for the data obtained in the experiment.Following a significant ANOVA result (p < 0.05), a post-hoc test, the Least Significant Difference (LSD) test, was employed to identify specific pairwise differences between treatment means.
Ammonium sulphate (21% N) led to moderate increases in leaf length and width with increasing N doses (Table 1).However, similar to the number of leaves, leaf length at the highest dose of 400 mg N kg −1 was lower compared to 200 mg N kg −1 N dose.The leaf length in SRAS treatments significantly increased with increasing N doses upto 200 mg N kg −1 N dose and remained constant at the highest N dose.The difference in leaf length response between AS and SRAS fertilizers suggest that the slow-release mechanism might have contributed to better nutrient uptake even at the highest N application dose.Calcium nitrate, with a N content of 11.8%, also showed a trend of increasing leaf length and width with higher N doses.The increase in leaf length and width was relatively consistent across all doses, indicating a steady response to N supplementation.Yeast residue, with a lower N content of 3% but a higher organic matter content of 35% and organic carbon content of 16%, resulted in variable effects on leaf length and width.While lower N doses showed smaller leaf lengths compared to other fertilizers, the highest N dose led to the longest (10.3 ± 0.5 cm) and widest (5.3 ± 0.2 cm) leaves observed in the experiment.
The ANOVA revealed a significant effect of both fertilizer type and N dose on plant height (P = 0.01).There was also a significant interaction effect between fertilizer type and N dose (P = 0.01).The highest dose of YR (YR×400) resulted in the tallest plants overall (23.7 cm), while lower YR doses had minimal impact (Table 1).The CN treatments generally produced taller plants compared to some AS or SRAS treatments, with CN×400 (20.5 cm) being the tallest among CN groups.AS and SRAS showed the most variation, with AS×200 achieving a height comparable to the tallest CN treatment, but other AS and SRAS doses resulting in shorter plants.

The effect of different fertilizer types and doses on fresh weight
Fresh weight of spinach plants at each storage period (0, 5, and 10 days) was significantly different from each other.The effect of fertilizer type on fresh weight was not statistically significant (P = 0.054).Additionally, significant differences (P = 0.01) were evident within each fertilizer type across the different N doses (Table 2).The effect of fertilizer type × N dose interaction on fresh weight was significant (P = 0.010), while Day × fertilizer type (P = 0.080), Day × N dose (P = 0.095) and Day × Fertilizer Type × N Dose (P = 0.752) interactions were not statistically significant.At the begining of storage period (day 0), the AS and CN fertilizers showed an increase in fresh weights of spinach with increasing N dose up to 200 mg kg −1 , followed by a decrease at 400 mg N kg −1 (Table 2).On Day 5, the highest yield in the AS application was Table 2 The effects of different N sources and doses on fresh weight of spinach plants during different storage days.Each cell in the table contains mean ± standard error of mean values for the fresh weights of spinach plants in each pot along with the letters for statistical analysis.7.8 ± 1.6 k-s 5.9 ± 0.9 o-s 7.4 ± 0.9 l-s 4.7 ± 0.3 rs 50 8.7 ± 2.3 I-s 8.3 ± 0.7 j-s 8.9 ± 0.3 I-s 7.3 ± 0.9 s 100 11.9 ± 3.1 a-m 12.2 ± 2.5 a-l 12.1 ± 1.5 a-l 9.0 ± 1.1 i-s 200 14.9 ± 0.9 a-f 11.2 ± 1.1 c-n 9.9 ± 1.3 f-r 10.9 ± 0.4 d-p 400 13.1 ± 1.4 a-j 13.9 ± 0.3 a-i 15.2 ± 3.5 a-e 14.3 ± 1.3 a-h Day 10 25 4.8 ± 2.2 qrs 4.1 ± 1.2 s 6.3 ± 0.6 n-s 4.1 ± 0.6 s 50 5.6 ± 1.0 p-s 6.6 ± 1.2 m-s 8.4 ± 2.4 j-s 6.2 ± 0.7 n-s 100 8.4 ± 2.4 j-s 5.2 ± 0.7 qrs 9.9 ± 1.9 f-r 8.0 ± 0.5 j-s 200 10.1 ± 1.9 e-p 10.9 ± 1.4 d-p 14.4 ± 1.1 a-h 7.6 ± 1.6 k-s 400 8.5 ± 1.7 j-s 14.7 ± 0.4 a-g 14.7 ± 3.0 a-g 16.9 ± 1. recorded at the 200 mg N kg −1 N dose (14.9 g pot −1 ), while the highest fresh spinach weights in other fertilizer types were obtained at the 400 mg N kg −1 N dose (Table 2).A similar pattern of weight increase and decrease with increasing N dose was observed in the AS application throughout all days.Weight increased up to the 200 mg N kg −1 N dose on all days but decreased at the 400 mg N kg −1 N dose.In SRAS, the slow-release form of AS, weight increased with increasing N dose up to 200 mg N kg −1 on Day 0, and there was a statistically insignificant decrease at the 400 mg N kg −1 N dose.However, on Day 5 and Day 10, weight consistently increased with increasing N dose.In YR, spinach fresh yield consistently increased with increasing N dose.The increase became more pronounced when increasing the N dose from 200 to 400 mg N kg −1 every days.The weight, initially 13.3 g on the Day 0, increased to 16.6, 10.9 g pot −1 on Day 5 increased to 14.3 g pot −1 , and the weight of 7.6 g pot −1 on Day 10 increased to 16.9 g pot −1 at the 400 mg N kg −1 N dose.

Period
In the CN application, a similar trend to AS was observed on Day 0, while on Day 5 and Day 10, an increase in fresh spinach yield was observed with increasing N dose.
The effect of different fertilizer types and doses on NO 3 -content of spinach plants doses, with AS and CN showing the most consistent response.This aligns with the findings of Özenç & Şenlikoğlu (2017) who reported a 12% increase in spinach leaf number with higher N doses, and Purquerio et al. (2007) who observed larger leaf area in arugula plants with higher N doses.These findings highlight the importance of both N content and organic matter characteristics when selecting fertilizers for for leafy crops.Plant length, a crucial morphological parameter indicating plant vigor and growth of plants increased with higher N doses across fertilizer types.The most pronounced increase was observed for CN treatment (44% increase at the highest N dose), possibly due to calcium's role in cell wall structure enhancement, as reported by Kacar & Katkat (2015).Additionally, reduced sodium uptake associated with CN fertilization, as observed by Ebert et al. (2002), may contribute to improved plant growth.This aligns with findings from Thapa et al. (2021), Zaman et al. (2018) and Shormin & Kibria (2018) who reported increased plant height and leaf number with high N application.The observed increase in plant growth parameters can likely be attributed to enhanced photosynthetic activity due to increased N availability (Kubar et al., 2022).
Table 3 The effects of different N sources and doses on nitrate concentration of spinach plants during different storage days.Each cell in the table contains mean ± standard error of mean values for the nitrate contents of spinach plants in each pot along with the letters for statistical analysis.Chlorophyll content indicated by SPAD values, a generally increased with higher N doses for AS, SRAS, and CN treatments, indicating improved chlorophyll systhesis leaf health.The YR treatment resulted in moderate increases in SPAD values, suggesting a less pronounced effect compared to AS, CN, and SRAS.The positive relationship between plant N nutrition and SPAD values observed in this study aligns with established knowledge (Esfahani et al., 2008;Hou et al. 2021).As reported by Porter & Evans (1998), increasing N concentration in leaves, associated with higher N application, enhances the intensity of light utilized during photosynthesis.However, while we did not directly measure photosynthesis, the positive relationship between SPAD values and N dose in our study suggests potentially improved photosynthetic activity, particularly for AS and SRAS treatments with the highest SPAD values.The observed variability in SPAD values across N fertilizer types suggests that fertilizer type, beyond just N content, may influence chlorophyll content and potentially photosynthetic performance.Future studies directly measuring photosynthesis are needed to confirm this hypothesis.Although Han et al. (2023) highlighted the complexity of NO − 3 stress on spinach leaves, our results generally support the positive relationship between N dose and SPAD values.Future studies should directly measure photosynthesis to confirm these findings and explore potential strategies for mitigating NO − 3 stress to ensure food safety.

Fresh weights of spinach plants during various storage periods
Numerous studies have highlighted the positive effects of N application and various N fertilizer types on plant growth and yield (Albayrak & Çamaş, 2006;Tekeli & Daşgan, 2013).Nitrogen serves a pivotal role in stimulating vegetative growth, enhancing leaf development, and improving overall plant health.Furthermore, N fertilization has been shown to enhance photosynthesis, leading to increased biomass production and improved crop yields (Züst & Agrawal, 2016).However, recent findings by Han et al. (2023) suggest a more nuanced relationship between NO − 3 levels and plant growth.Their study revealed that excessive NO − 3 concentrations could significantly reduce plant biomass, indicating negative effects on growth.Similarly, our observations indicated a decrease in spinach plant fresh weights at the highest N doses (400 mg kg −1 ) for all N fertilizer types, highlighting the importance of optimizing fertilization to avoid adverse effects on growth and yield.
While N application can enhance plant growth and yield, increasing it beyond a certain point can be counterproductive (The, Snyder & Tegeder, 2021).Our study, along with findings by Han et al. (2023), underscores the importance of optimizing N fertilization strategies.This is crucial to maximize plant growth and yield while minimizing negative environmental consequences, such as N emissions (Guo, Liu & He, 2022;Menegat, Ledo & Tirado, 2022).Our study also revealed differences in yield response among different N fertilizer types.YR exhibited a continuous yield increase, indicating a potentially slower N release compared to AS and CN, which showed a decrease at the highest N dose.SRAS shows a similar increase as AS up to moderate N dose (200 mg N kg −1 ), but without the sharp decline observed with AS and CN at the highest dose (400 mg N kg −1 ).This suggests a slower release profile for SRAS compared to AS, but without the negative impact of application of AS and nitrification inhibitors DCD and DMPP under field conditions resulted in lower NO − 3 concentrations in spinach plants.Slow released fertilizers are nitrification inhibitor fertilizers that inhibit the activity of nitrifying bacteria responsible for converting ammonium to nitrate, thereby allowing nitrogen to remain in the ammonium form for 4-8 weeks depending on soil conditions (Scheffer & Bartels, 1998).Similar to Güneş (2021), Montemurro et al. (2008) reported that the application of the nitrification inhibitor DCD fertilizer reduced NO − 3 concentration in lettuce by 24% compared to urea application.
Organic N sources like YR, consistently maintained lower NO − 3 levels in spinach plants, throughout the experiment, potentially minimizing the risk of excess NO − 3 accumulation compared to inorganic fertilizers.The slower release of N from organic source, which may not be fully utilized by the plants during experimental period, could contribute to this observation.This is because organic fertilizers typically do not provide N in a readily available form (Herencia et al., 2011).Therefore, NO − 3 accumulation in edible part of crops is usually lower in organically grown crops than in conventionally grown crops (Pavlou, Ehaliotis & Kavvadias, 2007).Supporting this finding, Liu et al. (2014) showed that lettuce grown under organic fertilizer (200 kg N ha −1 ) accumulated 14 to 19% less NO − 3 compared to mineral nitrogen fertilizer at (200 kg N ha −1 as NH 4 NO 3 ).

CONCLUSIONS
This study investigated the impact of nitrogen fertilizer type and application rate on the growth, nitrate content, and storage quality of spinach plants.The findings highlight the importance of tailoring nitrogen fertilization strategies to achieve desired outcomes.For producers seeking rapid early growth and potentially higher yields, ammonium sulphate (AS) emerged as a viable option at moderate doses (200 mg N kg −1 ).However, the observed decrease in growth at the highest dose (400 mg N kg −1 ) underscores the importance of careful monitoring to avoid exceeding safe nitrate limits set by regulatory bodies.
Slow-release ammonium sulphate (SRAS) presented a more balanced approach.While promoting sustained growth comparable to AS at moderate doses, SRAS resulted in generally lower peak nitrate concentrations, potentially reducing the risk of exceeding safe limits.This characteristic makes SRAS a promising option for producers seeking to optimize both yield and consumer safety.Yeast residue (YR) emerged as a viable option for organic spinach production.Despite its lower nitrogen content compared to other fertilizers, YR still promoted plant growth, albeit at a slower rate.Importantly, YR consistently maintained the lowest nitrate levels throughout the experiment, offering a potential solution for organic producers concerned about nitrate accumulation.
While the study provides valuable insights into the effects of different nitrogen fertilizers on spinach growth and nitrate content.However, it is essential to acknowledge some limitations that may affect the generalizability of the findings.The study was conducted under controlled greenhouse conditions.Real-world field conditions can vary significantly in terms of temperature, light intensity, rainfall patterns, and soil properties.These factors can influence plant growth, nutrient uptake, and nitrate accumulation.

Table 1
Impact of fertilizer type and nitrogen doses on leaf properties and SPAD values in spinach.Each cell in the table contains mean values ± standard error of mean for the leaf parameters and SPAD readings along with the letters for statistical analysis.